ABCMdb

PMID: 9374552

Fanen P, Labarthe R, Garnier F, Benharouga M, Goossens M, Edelman A
Cystic fibrosis phenotype associated with pancreatic insufficiency does not always reflect the cAMP-dependent chloride conductive pathway defect. Analysis of C225R-CFTR and R1066C-CFTR.
J Biol Chem. 1997 Nov 28;272(48):30563-6., [PubMed]

Sentences

No. Mutations Sentence Comment

2 ABCC7 p.Arg1066Cys ABCC7 p.Cys225Arg C225R and R1066C are both associated with pancreatic insufficiency, but the former mutation is associated with mild and unusual lung disease, whereas the latter is associated with severe lung disease. Login to comment 4 ABCC7 p.Arg117His ABCC7 p.Cys225Arg ABCC7 p.Cys225Arg Immunoprecipitation and functional studies showed that cells transfected with C225R-CFTR exhibit cAMP-dependent chloride fluxes; C225R-CFTR protein is poorly expressed but fully glycosylated and can be compared with R117H-CFTR. Login to comment 5 ABCC7 p.Arg1066Cys R1066C-CFTR protein is not correctly processed and, unlike ⌬F508-CFTR, this defect cannot be corrected by reduced temperature or overexpression in butyrate-treated cells; defective processing may occur at a different step in the biosynthetic pathway. Login to comment 13 ABCC7 p.Arg1066Cys ABCC7 p.Cys225Arg The first lies within the fourth putative membrane-spanning domain and the second within the fourth intracellular loop of the CFTR protein, replacing a cysteine by an arginine at position 225 and an arginine by a cysteine at position 1066, respectively (6). Login to comment 15 ABCC7 p.Cys225Arg ABCC7 p.Cys225Arg Immunoprecipitation and functional analysis using a halide- sensitive indicator showed that cells transfected with C225R-CFTR exhibit cAMP-dependent chloride fluxes; C225R-CFTR protein is poorly expressed but fully glycosylated. Login to comment 16 ABCC7 p.Arg1066Cys ABCC7 p.Arg1066Cys Cells transfected with R1066C-CFTR did not respond to cAMP stimulation, and R1066C-CFTR protein was not fully glycosylated, reflecting a defect in protein biosynthesis. Login to comment 17 ABCC7 p.Arg1066Cys Contrary to ⌬F508-CFTR, the defect in R1066C-CFTR transfected cells was not corrected by overexpression or a lower growth temperature. Login to comment 23 ABCC7 p.Arg117His ABCC7 p.Arg1066Cys ABCC7 p.Cys225Arg Three mutants, C225R, R1066C, and R117H, were constructed. Login to comment 57 ABCC7 p.Arg117His ABCC7 p.Arg1066Cys ABCC7 p.Cys225Arg RESULTS Three mutated CFTR proteins (C225R-, R1066C-, and R117H-CFTR) and wild-type CFTR were transiently expressed in HeLa cells and analyzed at the mRNA, protein, and functional levels. Login to comment 58 ABCC7 p.Arg117His Wild-type and R117H-CFTR were used as controls since both proteins are processed to the plasma membrane and function as cAMP-regulated chloride channels (3, 11). Login to comment 63 ABCC7 p.Arg117His ABCC7 p.Arg1066Cys ABCC7 p.Arg1066Cys ABCC7 p.Arg1066Cys ABCC7 p.Cys225Arg ABCC7 p.Cys225Arg A, Northern blot analysis; B, immunoprecipitation assay using mAb 24-1 (Genzyme) of mock-transfected HeLa cells expressing wild-type and mutant CFTRs without (left panel) and with (right panel) sodium butyrate treatment at the same exposure time; C, immunoprecipitation assay of R1066C- and ⌬F508-transfected cells grown at 26 or 37 °C. TABLE I Summary of the MEQ assay results Cell type Wild-type R117H C225R R1066C C225R ϩ NaBd R1066C ϩ NaBd pECE All responding 60 (30)a 9 (16)a 8 (8)a 0 11 (22)a 0 0 Fast 30 (50)b 3 (33)b 0 0 3 (27)b 0 0 ⌬Fstim/⌬Fbasal 13.5 Ϯ 6.8c 8 Ϯ 1.7c 22.3 Ϯ 7.1c Range 6-27 6-9 16-30 Slow 30 (50)b 6 (66)b 8 (100)b 0 8 (73)b 0 0 ⌬Fstim/⌬Fbasal 3.4 Ϯ 1.0c 2.4 Ϯ 0.5c 2.9 Ϯ 1.4c 3.4 Ϯ 1.0c Range 2-5 2-3 1.5-5 1.5-5 Total 200 56 100 100 50 50 60 a Percentage of all cells. Login to comment 66 ABCC7 p.Arg117His Fig. 1B shows that the fully processed form of wild-type CFTR protein was detected in HeLa cells as a diffuse band of approximate molecular mass of 170 kDa (band C) and that the core-glycosylated form appeared as a thin band of about 140 kDa (band B) on 5% SDS-PAGE; the pattern was almost identical for R117H-CFTR. Login to comment 67 ABCC7 p.Cys225Arg C225R-CFTR expression showed a faint band C or B, indicating that some mutant protein matured and reached the cell surface. Login to comment 68 ABCC7 p.Arg1066Cys R1066C-CFTR produced a low level of core-glycosylated CFTR as a faint band B, suggesting a low level of synthesis and defective biosynthesis (Fig. 1B). Login to comment 70 ABCC7 p.Arg117His ABCC7 p.Arg117His ABCC7 p.Cys225Arg ABCC7 p.Cys225Arg The intensity of bands C and B of wild-type, R117H- and C225R-CFTR increased markedly, and the ratio of bands C/B was similar in treated wild-type, R117H-, and C225R-CFTR cells. Login to comment 71 ABCC7 p.Arg1066Cys Although synthesis of R1066C-CFTR increased markedly with sodium butyrate (strong band B), we did not detect a mature band C, suggesting that this mutant cannot function as a cAMP-regulated chloride channel (Fig. 1B). Login to comment 72 ABCC7 p.Arg1066Cys To determine if R1066C was a temperature-sensitive mutant of CFTR, cells were grown at 24-26 °C for 48 h and harvested for immunoprecipitation-PKA assay; ⌬F508 was used as control as it has been shown that a reduced growth temperature permits maturation and delivery to the plasma membrane (13). Login to comment 73 ABCC7 p.Arg1066Cys No fully glycosylated CFTR (band C) protein was detected when R1066C-CFTR transfected cells were grown at 24-26 or 37 °C, whereas band C appeared when ⌬F508-CFTR cells were grown at 24-26 °C (Fig. 1C). Login to comment 74 ABCC7 p.Arg1066Cys Thus, neither butyrate treatment nor low temperature rescued R1066C-CFTR. Login to comment 80 ABCC7 p.Arg117His ABCC7 p.Arg117His Similar results were obtained with R117H-transfected cells (33% being fast responsive and 66% slow responsive cells) supporting the immunoprecipitation data, which suggested that wild-type and R117H-CFTR activate a cAMP-regulated chloride pathway. Login to comment 81 ABCC7 p.Cys225Arg When exposed to the stimulatory mixture, 8% of C225R-CFTR cells showed an increased rate of change in MEQ fluorescence, indicating the activation of a cAMP-dependent anion pathway. Login to comment 83 ABCC7 p.Arg1066Cys None of the R1066C-CFTR cells (n ϭ 100) exhibited an increase in MEQ fluorescence when exposed to the stimulatory mixture (Fig. 2C), supporting the notion that only fully glycosylated CFTR is functional. Login to comment 84 ABCC7 p.Arg117His ABCC7 p.Arg1066Cys ABCC7 p.Cys225Arg To determine if increased protein synthesis influenced the function of the C225R and R1066C mutants, we pretreated cells with 5 mM sodium butyrate for 18-20 h. R117H- and wild-type-CFTR were not tested since they were fully responsive in untreated conditions. Login to comment 85 ABCC7 p.Cys225Arg After treatment with butyrate, 22% of C225R-CFTR cells were responsive; of these, 27 and 73% were fast and slow responsive, respectively (Table I). Login to comment 87 ABCC7 p.Cys225Arg These results strengthen the conclusion that C225R-CFTR protein is correctly processed to the plasma membrane. Login to comment 88 ABCC7 p.Arg1066Cys Sodium butyrate treatment of R1066C-CFTR cells (n ϭ 50) induced no change in MEQ fluorescence, indicating that butyrate treatment cannot overcome the defective function of this mutant. Login to comment 93 ABCC7 p.Arg117His ABCC7 p.Arg117His A, HeLa cells transfected with wild-type (WT) CFTR, R117H-CFTR, or pECE (⌬Fstim/⌬Fbasal ϭ 21 for WT fast, 2 for WT slow, 6 for R117H, and 1 for pECE). Login to comment 94 ABCC7 p.Cys225Arg ABCC7 p.Cys225Arg B, HeLa cells transfected with C225R-CFTR with and without sodium butyrate (NaB) treatment (⌬Fstim/⌬Fbasal ϭ 22 and 1.5 for C225R with and without sodium butyrate, respectively). Login to comment 95 ABCC7 p.Arg1066Cys C, HeLa cells transfected with R1066C-CFTR with sodium butyrate treatment (⌬Fstim/⌬Fbasal ϭ 1). Login to comment 96 ABCC7 p.Arg117His ABCC7 p.Arg1066Cys ABCC7 p.Cys225Arg CFTR Mutations Associated with Pancreatic Insufficiency atUniversityofNorthCarolinaatChapelHill,onOctober25,2012www.jbc.orgDownloadedfrom DISCUSSION We analyzed the structure-function relationships of two mutations, C225R and R1066C, that we had identified in CF patients with pancreatic insufficiency (6) and compared the properties of those mutations with those of wild-type-CFTR and R117H-CFTR. Login to comment 97 ABCC7 p.Cys225Arg C225R was found in a compound heterozygote for the ⌬F508 mutation and was associated with pancreatic insufficiency, normal lung function, and asthma. Login to comment 98 ABCC7 p.Arg1066Cys R1066C was first found in a patient bearing the ⌬F508 mutation on the other chromosome. Login to comment 99 ABCC7 p.Arg1066Cys Six unrelated patients bearing the R1066C mutation have since been identified. Login to comment 100 ABCC7 p.Asp110His Five patients had pancreatic insufficiency while the sixth had normal pancreatic function and was a compound heterozygote for a mild mutation (D110H) (14). Login to comment 101 ABCC7 p.Arg117His ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro ABCC7 p.Arg347His ABCC7 p.Pro99Leu ABCC7 p.Pro205Ser Six CF-associated mutations (P99L, R117H, P205S, R334W, R347P, and R347H) located in putative membrane-spanning domains that have already been analyzed for their functional properties (2-5) were all associated with a mild phenotype (pancreatic sufficiency, PS). Login to comment 102 ABCC7 p.Cys225Arg To our knowledge, this is the first reported expression of the C225R-CFTR mutant and the first description of a transmembrane mutant associated with a severe phenotype (PI). Login to comment 103 ABCC7 p.Cys225Arg Heterologous expression of this mutant in HeLa cells showed that C225R-CFTR protein elicited cAMP-dependent chloride fluxes. Login to comment 104 ABCC7 p.Cys225Arg It thus seems that the pancreatic insufficiency associated with the C225R mutation cannot be fully explained by defective chloride conduction through the mutant CFTR protein. Login to comment 105 ABCC7 p.Arg117His ABCC7 p.Cys225Arg The processing of C225R-CFTR resembles that of R117H-CFTR. Login to comment 107 ABCC7 p.Arg117His ABCC7 p.Cys225Arg Both mutants are associated with mild lung disease, but R117H is associated with PS whereas C225R is associated with PI. Login to comment 109 ABCC7 p.Arg1066Cys Regarding the second mutant studied here, R1066C-CFTR, no cAMP-activated anion conductance was found in cells expressing this protein and no mature protein was detected. Login to comment 111 ABCC7 p.Arg1066Cys During the course of this study, heterologous expression of R1066C-CFTR was reported by others using different expression systems (15, 16). Login to comment 112 ABCC7 p.Arg1066Cys Our results are in keeping with the report by Seibert et al. R1066C is a class II mutation in the classification proposed by Welsh and Smith (17). Login to comment 114 ABCC7 p.Arg1066Cys Unlike ⌬F508-CFTR, R1066C-CFTR protein cannot bypass the "quality control" of the endoplasmic reticulum when grown at reduced temperature or when overexpressed in butyrate-treated cells; defective processing may thus occur at a different step in the biosynthetic pathway. Login to comment 117 ABCC7 p.Arg1066Cys ABCC7 p.Cys225Arg At the molecular level, cysteine replacement, as in C225R and R1066C, may lead to the disruption or creation of disulfide bonds between cysteines and thereby change the channel properties. Login to comment